Stractural and molecular biological study of the ciliary basal apparatus during ciliogenesis

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1. We obtained six monoclonal antibodies that labeled striated rootlets associated with basal bodies extending cilia. These antibodies identified proteins of 205-215 kDa by immunoblotting. Using a clone R67, we revealed the complicated morphology of striated rootlets associated with solitary cilia. The 205-215 antigens were constitutive components of dark striations of the rootlet. Striated rootlets were positively stained in all cell types in the human submandibular glands. It is suggested that striated rootlets are concerned with the positioning of centrioles and may be associated with centrioles under normal physiological conditions, without formation of solitary cilia.2. Treatment with colcemid resulted in the elongation and/or structural complication of the centriole-associated rootlets in PtK_2 cells and additionally, organization of intracytoplasmic free rootlets. The changes were reversible and almost disappeared 2 hours after removal of the drug. These observations may indicate functional relationships between cytoplasmic microtubules and striated rootlets, and also the existence of rootlet-nucleating factors in the cytoplasm in addition to centrioles.3. The localization of gamma-tubulin in the ciliary basal apparatus was studied by fluorescence immunohistochemistry and immunoelectron microscopy. Ip addition to basal bodies, gamma-tubulin was identified in the lateral basal foot, especially the basal foot cap, confirming the previous observations that the basal foot serves as the microtubule organizing center.4. In the cells extending solitary cilia, the basal body and the single centriole were interconnected by one or two bundles of thin filaments with a few periodic striations. We have called these bundles the striated connector. The connector could play an important role for stabilising the basal body in the apical cytoplasm.